Chromatography is a widely used laboratory technique for the separation of components in a mixture based on their differential interactions with the stationary phase. This technique aids in the identification and purification of components and is applicable for both qualitative and quantitative analysis.
Affinity chromatography is a type of liquid chromatography, which utilizes the reversible biological interaction (affinity) between components in the mobile phase and a solid stationary phase for separation.
- It was discovered by Pedro Cuatrecasas and Meir Wilcheck in 1968.
- Affinity chromatography falls under the broader category of adsorption chromatography. However, it is highly specific and selective in comparison to adsorption chromatography.
- In affinity chromatography, the adsorption process is elective and specific, relying on the binding affinity between target biomolecules and a ligand immobilized on the solid support matrix.
- It enables the purification of target biomolecules (often a protein) based on their biological structure and function.
- Some ligand-target interactions include enzyme-substrate, enzyme-inhibitor, antibody-antigen, lectin-glycoprotein, etc.
- Ligands obtained from biological sources include antibodies, enzymes, proteins, glycoproteins, and others. Synthetic ligands used are metal chelates, boronates, biomimetic dyes, etc.
- Prepacked columns such as HiTrap, HiPrep, etc., are commercially available, facilitating rapid optimization.
Principle of Affinity Chromatography
The principle of affinity chromatography is primarily based on the ability of a biologically active molecule to bind specifically and reversibly to a ligand immobilized to the surface of the solid stationary phase. The working mechanism of affinity chromatography is often associated with the lock-and-key mechanism of enzyme-substrate interaction. Here, the lock is the immobilized ligand on the chromatographic support, which is opened by the specific site on the target molecules, or protein, which serves as the key.
- The active binding sites of the ligand should be accessible to the components of the mixture for easy binding, and the binding sites should not be deformed during the immobilization of the ligand to the solid support.
- Substances that do not possess a complementary binding site for the ligand are eluted faster either directly through the column or by applying a low stringency washing step. Meanwhile, bound substances are recovered by flowing a suitable solvent through the column that breaks the affinity between bound substances and the ligand.
- The bound target molecules can be eluted by methods such as including a competing ligand in the mobile phase or changing the pH, ionic strength, or polarity conditions.
- Biological interactions between the target molecules and ligands result from electrostatic or hydrophobic interactions, Van der Waal’s forces, and hydrogen bonding. The interaction is reversed to elute the desired component either specifically using a more competitive ligand or non-specifically by altering the pH, polarity, or ionic strength.
- Affinity chromatography can proceed in a single-step or a multi-step process. The single-step procedure saves time and is more selective compared to multistep procedures. The concentrating effect in a single step enables large volumes to be processed.
- For a higher degree of purity, or in the unavailability of a suitable ligand for affinity purification, an efficient multi-step process must be developed using the purification strategy of Capture, Intermediate Purification, and Polishing (Cipp). When applying this strategy affinity chromatography offers an ideal capture or intermediate step in any purification protocol and can be used whenever a suitable ligand is available for the protein of interest.
Components of Affinity Chromatography
1. Matrix
- Chemically and physically inert support that directly or indirectly couples a ligand.
- Matrix should have a relatively larger surface area for better ligand attachment.
- It must be insoluble in solvents and buffers used in the purification process.
- It must exhibit good flow properties.
- Examples: agarose, polyacrylamide, polystyrene beads, cellulose, etc.
2. Spacer arm
- It connects the ligand to the matrix.
- Employed to improve binding efficiency between the target molecules and ligands by overcoming steric hindrances.
- Example: 1, 6-diaminohexance, 6-aminohexanoic acid and 1, 4-bis-(2,3-epoxypropoxy) butane.
3. Ligand
- Ligand refers to the molecule attached or immobilized to the matrix, which reversibly binds to a specific target molecule.
- The selection of a specific ligand depends on the nature of the target molecules.
- For example: antigen is used as a ligand for antibody isolation, substrate or inhibiter is employed as a ligand for isolation of enzyme, etc.
Ligands are mainly classified into two types: mono-specific and group-specific low molecular weight ligands.
- Monospecific low molecular weight ligands are the type of ligands that bind to a single or a small number of proteins or target molecules. For example: enzyme inhibitors, steroid hormones, vitamins, etc.
- Group-specific ligands bind to a large number of target molecules. It includes biomimetic dyes, boronic acid derivatives, 5’ AMP, etc.
Table: List of target proteins with their appropriate ligand.
| Ligand | Protein of interest |
| Enzyme | Substrate, inhibitor, cofactor |
| Antibody | Antigen, virus, cell |
| Lectin | Polysaccharide, glycoprotein, cell surface receptor, cell |
| Nucleic acid | Complementary base sequence, histones, nucleic acid polymerase, nucleic acid binding protein |
| Hormone, vitamin | Receptor, carrier protein |
| Glutathione | Glutathione-S-transferase or GST fusion proteins |
| Metal ions | Poly (His) fusion proteins, native proteins with histidine, cysteine and/or tryptophan residues on their surfaces |
| Protein A and Protein G | Immunoglobulins |
| Phenyl boronate | Glycoproteins |
| Poly (A) | RNA containing poly(U) sequences, RNA specific proteins |
| Nucleotides (5’ AMP, 2’5’-ADP) | NAD+ dependent dehydrogenase, NADP+ dependent dehydrogenase |
| Fatty acids | Fatty acids binding proteins |
Types of Affinity Chromatography
1. Boronate and Phenyl Borate Affinity Chromatography
- Boronate is used as an affinity ligand in the analysis of hemoglobin A1c (HbA1c) which is the component of glycated hemoglobin found in human blood.
- Cellufine phenyl borate is an affinity ligand used to purify glycoproteins, glycated protein, and diol compounds.
2. Lectin Affinity Chromatography
- Lectin is used as a stationary phase. Lectins are non-immune proteins that recognize and bind certain types of carbohydrate residues.
- Used to separate polysaccharides, glycopeptides, oligosaccharides, and cells that contain particular carbohydrate structures.
3. Dye-ligand Affinity chromatography
- Used to purify blood proteins, protein pharmaceutical agents, enzymes, and albumin.
4. Immunoaffinity chromatography
- Utilizes antibodies to purify peptides, viruses, hormones, and enzymes.
5. Immobilized Metal Ion Affinity Chromatography
- Interaction between immobilized metal ions and target amino acids, proteins, peptides, and nucleic acids. Metal ions are immobilized using chelating agents such as iminodiacetic acid, nitrilotriacetic acid, L-glutamic acid, etc.
- A powerful tool for analyzing membrane proteins, histidine-tagged proteins, and phosphorylated proteins.
- Used in sample pretreatment to detect drugs including tetracycline, quinolones, macrolides, β-lactams, and aminoglycosides.
- It has been used in the detection of biomarkers for disease diagnosis. This process involves the use of immobilized metal ion affinity chromatography with mass spectrometry in surface-enhanced laser desorption/ionization (SELDI).
6. Analytical Affinity Chromatography
- It is also referred to as quantitative affinity chromatography.
- Utilized for isolating and measuring specific targets.
Sample preparation for Affinity Chromatography
Samples should be clear and free from any particulate matter. Filtration, precipitation, and centrifugation processes can be utilized for sample clarification. Simple steps to clarify a sample before beginning purification will avoid clogging the column, may reduce the need for stringent washing procedures, and can extend the life of the chromatographic medium. The sample buffer should have optimal pH, and ionic strength, and should be compatible with the target molecules and ligands.
Any components known to disrupt the interaction should be removed. Contaminants are removed using detergents, reducing agents, protease inhibitors, and DNase or RNase.
Procedure or Steps of Affinity Chromatography
1. Preparation of column
- The affinity medium is first equilibrated in a binding buffer.
- The column is taken and loaded with appropriate solid support.
- The ligand is selected according to the nature of the target molecules and is bound to the solid support.
- The spacer arm is attached between the ligand and the solid support.
2. Loading of the sample
- A solution containing a mixture of the substances is poured into the column and allowed to flow through it at a controlled rate.
- The sample is applied in conditions that favor the specific binding of the target molecules to the complementary binding ligand.
3. Elution
- Target substances bind specifically, but reversibly, to the ligand and unbound material washes through the column.
- Elution is performed specifically, using a competitive ligand, or non-specifically, by changing the pH, ionic strength, and polarity, or using chaotropic eluents. The target protein is collected in a purified, concentrated form.
- Selective elution is applied in combination with group-specific ligands whereas; non-selective elution methods are used in combination with highly specific ligands.
- Affinity medium is re-equilibrated.
Factors Affecting Affinity Chromatography
- Specificity of Ligand: The ligand must be specific for the target molecule. Non-specific interactions can lead to poor separation.
- Affinity or binding strength of ligand: Optimal binding occurs when the interaction is neither too weak nor too strong. Low affinity results in poor yields since the target molecules might leak from the column or are washed away. High affinity also results in lower yields as target molecules may not dissociate from the ligand during elution.
- Nature of matrix: A physically and chemically inert matrix having a higher surface area is preferred. The pore size should also be appropriate for the target molecules.
- pH: Optimal pH ensures proper binding of the ligand and target. Extreme pH can denature proteins and disrupt or weaken the interactions.
- Nature of buffer used: The buffer should maintain pH, ionic strength, and stability for both ligand and target molecule.
- Flow rate- Optimal flow rate balances binding efficiency and processing time.
Common Products and Manufacturers of Affinity Chromatography
Table: Commercially available products and their manufacturers
| Name of Manufacturer | Common products |
| GE Healthcare (Cytiva) | CNBr-activated Sepharose 4B Activated CH-Sepharose 4BHiTrapTM and HiPrepTM columns |
| Pierce | Reacti-Gel (GX, HW-65F, 25DF, GF-2000) |
| Biosepra | Act-Ultrogel ACA 22 |
| BioRad | AffiGel 10 Gel, AffiGel 15 Gel ProfinityTM IMAC Resins |
| Merck | AF-CDI 650 Fractogel TSK |
Applications of Affinity Chromatography
- Purification of proteins and enzymes.
- Separation of active biomolecules from denatured and functionally differed forms.
- Isolation of pure substances present in low concentration in the crude sample.
- Used as an intermediate step in the purification process.
- Purification of monoclonal antibodies.
Advantages of Affinity Chromatography
- High specificity and selectivity.
- Molecules can be obtained in highly purified form in high yield.
- Reusable matrix
- Effective purification based on biological structure and functions.
- High recovery of the active compounds.
- Independent of the sample volume provided, the ligand is highly specific.
Limitations of Affinity Chromatography
- A time-consuming and complicated method.
- Limited availability and costly ligands. More solvents are required, which may be expensive.
- Labor-intensive procedures.
- Non-specific adsorption cannot be totally eliminated, and it can only be minimized.
- Proteins get denatured if the required pH is not adjusted.
Troubleshooting and Safety Considerations
- The column should be clean.
- Sample preparation techniques should be improved.
- Small bubbles in the column should be removed by passing a de-gassed buffer.
- The sample should be filtered.
- Fresh samples should be used since the sample might alter during storage.
- The column should be equilibrated in the binding buffer.
- Microbial growth rarely occurs in the column during use. However, in case of microbial growth in packed columns, storage of columns in 20% ethanol when possible is employed.
- Non-specific binding of the ligand and target molecules is avoided by using competitive ligands and maintaining optimal conditions of the buffer.
Recent advances and Innovations in Affinity Chromatography
- Development of support materials such as flow-through beads, magnetic beads and monolithic materials
- Development of novel ligands with a variety of interesting biological properties such as Adapter immunoaffinity chromatography, nanobody and recombinant ligands, etc
- Development of miniaturized chromatographic systems.
- Integration of automated systems for precise monitoring and control of conditions.
Conclusion
Affinity chromatography is an indispensable technique for the highly specific purification of biomolecules. Its ability to isolate target proteins based on biological interactions makes it essential for biochemical and pharmaceutical applications.
References
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